Construct comprising recognition domain of antibody against von willebrand factor-specific cleaving enzyme

ABSTRACT

The present invention provides an epitope recognized by an antibody (hereinafter, also referred to as an anti-ADAMTS-13 antibody) against a cleaving protease (hereinafter, also referred to as ADAMTS-13) specific to von Willebrand factor (hereinafter, also referred to as vWF), and a polypeptide comprising the epitope region. The present invention also provides a polypeptide located in a region from position 449 to position 687 in an amino acid sequence composing the ADAMTS-13, which is recognized by the anti-ADAMTS-13 antibody, or a peptide fragment derived from the polypeptide.

TECHNICAL FIELD

The present invention relates to the field of ethical drugs.Specifically, the present invention relates to an epitope recognized byan antibody (hereinafter, also referred to as an anti-ADAMTS-13antibody) against cleaving protease (hereinafter, also referred to asADAMTS-13) specific to von Willebrand factor (hereinafter, also referredto as vWF) involved in blood coagulation, and to a polypeptidecomprising the epitope region. The present invention is also relates toan antibody that recognizes the polypeptide.

The polypeptide or a peptide fragment thereof provided by the presentinvention, which comprises an epitope region recognized by an antibodyagainst ADAMTS-13 opens up the possibility of diagnosis given on thepresence or absence of an autoantibody against ADAMTS-13 or an absorbingagent for the autoantibody or ADAMTS-13 replacement therapy for apatient with diseases associated with positivity for the autoantibody.

BACKGROUND ART

vWF is a blood coagulation factor that is produced in a vascularendothelial cell and a megakaryocyte and is present as a multimerstructure (with a molecular weight of 500 to 20,000 kDa) where singlesubunits each composed of 2050 amino acid residues (approximately250-kDa monomers) are bound through a S—S bond. The concentration of vWFin blood is approximately 10 μg/ml, and in general, vWF with a highermolecular weight has higher specific activity.

The vWF has two major functions as a blood coagulation factor, one ofwhich is a function as a carrier protein that binds to and therebystabilizes blood coagulation factor VIII and another of which is afunction of helping platelets adhere and aggregate to the tissue beneathvascular endothelial cells of injured vascular walls to form plateletthrombi.

Thrombotic thrombocytopenic purpura (hereinafter, also referred to asTTP) is a disease that causes platelet thrombi in body tissue arteriolesand capillary vessels in the whole body. In spite of the currentprogression of medical technology, the mortality associated with thedisease has increased approximately threefold from 1971 to 1991.Pathologically, the TTP is considered to be caused by vascularendothelia cell injury and platelet aggregation in blood vessels.Immunohistologically, the presence of vWF in large amounts is observedin generated platelet thrombi, and the vWF is considered to play acrucial role in the pathogenesis of the disease. TTP is broadly dividedinto familial (congenital) TTP likely to have an inheritance factor andacquired (idiopathic) TTP developed especially in adults. Normal orhigh-molecular-weight vWF multimer structures are dominant in TTPpatients. Especially, unusually large vWF multimer (ULvWFM) and largevWF multimer (LvWFM) are presumed to play a crucial role in thepromotion of platelet aggregation and microthrombus formation under highshearing stress. On the other hand, vWF has been known to undergodigestion at the location between 842Tyr and 843Met by the action ofvWF-cleaving protease under high shearing stress in circulating blood ofhealthy individuals. Thus, a probable scenario of how TTP is caused isas follows: the activity of the protease in plasma is reduced for somereason and ULvWFM or LvWFM is increased to accelerate plateletaggregation, followed by platelet thrombus formation in the bloodvessel.

In 2001, a gene encoding vWF-cleaving protease also known as ADAMTS-13that is an active body having the activity of the protease describedabove was cloned by the present inventors (JP Patent Publication (Kokai)No. 2003-284570). The findings about the molecular structure ofADAMTS-13 are summarized below. The location of a residue number that isnumbered from methionine encoded by an initiation codon (ATG) is shownas a rough guide within parentheses (see SEQ ID No. 1).

The domain structure of ADAMTS-13 has a signal peptide preceding apropeptide that ends in a RQRR sequence as a furin cleavage motif,followed by a metalloprotease domain containing a reprolysin-typezinc-chelating region consisting of HEXXHXXGXXHD as a consensus sequence(to amino acid residue No. 284 (p285X)); via a disintegrin-like domainas found in snake venom metalloprotease (to amino acid residue No. 386(W387X)), there exists a first Tspl motif (Tspl-1) consisting ofapproximately 50 to 60 residues generally considered to be important formolecular recognition (to amino acid residue No. 448 (Q449X)), whichcontinues to a Cys-rich region containing a RGDS sequence as one of celladhesion motifs (to amino acid residue No. 580 (T581X)); and, through aspacer domain consisting of approximately 130 amino acid residues withno cysteine residue (to amino acid residue No. 687 (W688X)), additionalTspl motif repeats (Tspl-2 to Tspl-8) follow, which is followed by a CUBdomain-1, and -2 that are said to be first found in a complementcomponent C1r or C1s.

By the way, no finding about a major neutralizing epitope region inADAMTS-13 has been obtained so far. In addition, a convenient diagnosticmethod for a patient positive for an autoantibody against the proteasehas not been established.

In light of such circumstances, a first object of the present inventionis directed to an invention relating to the identification of aneutralizing epitope present on ADAMTS-13 and a neutralizing/absorbingagent for an antibody thereby proposed, which is mainly intended for anautoantibody.

A second object of the present invention is to provide a method ofproducing the neutralizing/absorbing agent.

A third object of the present invention is to provide an application ofthe neutralizing/absorbing agent.

A fourth object of the present invention is to provide a method ofproducing a full-length or partially modified molecule of vWF-specificcleaving protease, which is obtained by modifying the epitope.

A fifth object of the present invention is to provide an application ofthe full-length or partially modified molecule of vWF-specific cleavingprotease, which is obtained by modifying the epitope.

Up to now, plasmapheresis has been provided as treatment to a patientcongenitally deficient in vWF-specific cleaving protease and a patientwith acquired production of an antibody against the protease. Therefore,there is a demand for the establishment of replacement therapy with purevWF-specific cleaving protease such as the protease purified orgenetically altered. It has been reported that a familial TTP patient iscongenitally deficient in vWF-specific cleaving protease andnon-familial TTP is caused by the acquired production of an autoantibodyagainst the protease. Thus, replacement therapy with the protease ispreferred for a familial TTP patient (in reality, plasma administrationis given to the patient), while a non-familial TTP patient requires theremoval of an autoantibody by plasma exchange as well as thesupplementation of the protease.

However, in the administration of ADAMTS-13 for supplementation to apatient positive for an autoantibody, an antibody against the protease,that is, an autoantibody, present in blood of the patient neutralizesthe administered protease. As a result, the protease loses enzymeactivity and has substantial reduction in concentration. However, theuse of a neutralizing region identified by a method of determining anepitope for an antibody against ADAMTS-13 disclosed in a previousapplication (JP Patent Application No. 2002-279924) or identified by thepresent invention as well as the preparation of a partially modifiedmolecule of a neutralizing epitope region that can be newly identifiedby Western blotting of competitive inhibition assay used in the presentinvention allows the administration of the protease to a patientpositive for an antibody against the protease; or alternatively, allowsthe absorption of the antibody by a polypeptide or the like containingthe neutralizing region provided by the present invention.

DISCLOSURE OF THE INVENTION

As a result of conducting diligent studies for attaining the isolationand identification of vWF-cleaving protease under the above-describedcircumstances, the present inventors successfully purified and isolatedheretofore unreported vWF-cleaving protease of interest and identifiedan amino acid sequence of a mature protein thereof and a gene encodingthe amino acid sequence in the previous application (JP PatentPublication (Kokai) No. 2003-284570).

Based on the findings obtained using genetic recombination techniquesdescribed in the previous application (JP Patent Publication (Kokai) No.2003-284570), the present inventors identified a region likely to beessential for activity expression (JP Patent Application No.2002-279924). From the results of using a mutant molecule prepared onthe basis of these findings to analyze a major neutralizing regionrecognized by an autoantibody against anti-ADAMTS-13 in an acquired TTPpatient in the present invention, it has been revealed that the regionrecognized by the autoantibody is in agreement with the above-describedregion likely to be essential for activity expression and is located ina region from a Cys-rich region (at approximately position 499) to aspacer region (at approximately position 687). Accordingly, a principalrequirement of a major neutralizing epitope region provided by thepresent invention for an anti-ADAMTS-13 antibody is the region from theCys-rich region (at approximately position 499) to the spacer region (atapproximately position 687) in a polypeptide composing ADAMTS-13 or apeptide fragment having an equivalent amino acid sequence. That is, thepresent invention relates to a polypeptide comprising a neutralizingepitope region in von Willebrand factor-specific cleaving protease(hereinafter, also referred to as vWFCP or ADAMTS-13), which isrecognized by an antibody against the protease, or a peptide fragmentderived from the polypeptide. The neutralizing epitope region in thepolypeptide or the peptide fragment derived from the polypeptide that isclaimed in claim 1 is located in a region from position 449 to position687 in an amino acid sequence shown in SEQ ID No. 1. The presentinvention is further intended to a polypeptide comprising an amino acidsequence from position 449 to position 687 in an amino acid sequenceshown in SEQ ID No. 1, or a peptide fragment derived from thepolypeptide. The present invention is also intended to a polypeptidecomprising an amino acid sequence from position 449 to position 687 inan amino acid sequence shown in SEQ ID No. 1 where one or several aminoacids are deleted, substituted, or added, the polypeptide beingrecognized by an antibody against von Willebrand factor-specificcleaving protease, or a peptide fragment derived from the polypeptide.One or several amino acids used herein refers to one to five aminoacids, preferably one to three amino acids, more preferably one or twoamino acids.

Using, as an antigen, a polypeptide or the like of the neutralizingepitope region, which is prepared on the basis of the amino acidsequence of ADAMTS-13 obtained by this finding, for example, monoclonaland polyclonal antibodies can be created by a typical immunizationmethod (Current Protocols in Molecular Biology, edited by F. M. Ausbelet al., (1987); Antibody Engineering: A PRACTICAL APPROACH, edited by J.McCAFFERTY et al., (1996); Antibodies: A Laboratory Manual, edited byHarlow David Lane (1988); or ANTIBODY ENGINEERING second edition, editedby Carl A. K. BORREBAECK (1995)). Alternatively, an antibody binding tothe protein (ADAMTS-13) can be created by a technique of creating anantibody by use of a phage display technique (Phage Display of Peptidesand Proteins: A Laboratory Manual, edited by Brian K. Kay et al.,(1996); Antibody Engineering: A PRACTICAL APPROACH, edited by J.McCAFFERTY et al., (1996); or ANTIBODY ENGINEERING second edition,edited by Carl A. K. BORREBAECK (1995)). Based on these techniques, aneutralizing antibody for the activity of the protease of the presentinvention or an antibody simply binding to the protease can also beisolated from a sample from a TTP patient positive for an autoantibodyagainst the protease of the present invention. In addition, the use ofthese antibodies allows the application of the present invention todiagnosis and treatment for a disease associated with variations in theamount of the protease of the present invention, for example, TTP. Thepresent invention also encompasses these antibodies.

In one embodiment, the present invention relates to a method ofdiagnosing a patient with a TTP-like disease or a patient at risk ofdeveloping vWF-dependent thrombosis, and the method comprises stepsbelow.

Diagnostic assay for a disease associated with variations in the amountof the protease of the present invention is performed using a biologicalsample from the patient. Such a sample can be used directly in the assayor, in some cases, can need to be subjected prior to the assay totreatment such as the removal of possible interfering substances in thesample. Examples of suitable biological samples include blood, urine,sweat, tissue, or serum. The method involves detecting an autoantibodyagainst vWF-cleaving protease in the biological sample. The steps of themethod are as follows:

(a) bringing a biological sample obtained from the patient into contactwith a solid support in which ADAMTS-13 or a partial peptide fragmentthereof is immobilized;

(b) bringing the solid support into contact with an anti-humanimmunoglobulin antibody labeled with a developer; and

(c) detecting the label of the specifically bound developer in the step(b) in order to obtain a value corresponding to the concentration of ananti-ADAMTS-13 antibody in the sample.

The above-described diagnosis can be conducted by immunoassay known inthe art. Solid supports that can be used include beads and plates madeof a resin such as polystyrene. Developers that can be used includeradioisotopes, enzymes such as peroxidase and alkaline phosphatase, andfluorescent substances.

In an alternative embodiment of the present invention, the polypeptideof the present invention is also useful as a neutralizing agent for anautoantibody by administering the polypeptide to a patient positive foran anti-ADAMTS-13 antibody or as a removing agent for an autoantibody.In this case, the neutralization of the autoantibody refers to bindingto the autoantibody, thereby inhibiting the binding of the autoantibodyto vWF-cleaving protease. In this method, the polypeptide is optionallyimmobilized in a suitable support or the like using a method known inthe art. Subsequently, a sample containing an anti-ADAMTS-13 antibody tobe removed, for example, blood from the patient is brought into contactwith the immobilized polypeptide to thereby remove the autoantibody fromthe sample from the patient. On this occasion, a carrier bound with aligand specific to the anti-ADAMTS-13 antibody is brought into contactwith blood or plasma from the patient and the anti-ADAMTS-13 antibody inthe blood or the plasma is bound to the ligand to thereby remove theantibody from the blood or the plasma. Subsequently, the blood or theplasma from which the antibody have been removed may be reinjected tothe patient. The above-described polypeptide or peptide fragment derivedfrom the polypeptide can be used as the ligand specific to theanti-ADAMTS-13 antibody. The contact may be performed, for example, byallowing the blood or plasma from the patient to pass through thecarrier bound with the ligand. The present invention further encompassesa method of producing blood or plasma free from an anti-ADAMTS 13antibody by bringing a carrier bound with the above-describedpolypeptide or peptide fragment derived from the polypeptide intocontact with blood or plasma from a patient positive for ananti-ADAMTS-13 antibody and binding an anti-ADAMTS-13 antibody in theblood or the plasma to the ligand to remove the antibody from the bloodor the plasma.

A neutralizing agent for an autoantibody administered to a patientpositive for an anti-ADAMTS-13 antibody is a pharmaceutical compositionfor treating a patient positive for an anti-ADAMTS-13 antibodycomprising, as an active ingredient, the above-described polypeptide orpeptide fragment derived from the polypeptide. The pharmaceuticalcomposition for treating a patient positive for an anti-ADAMTS-13antibody also includes a pharmaceutical composition for treating apatient positive for an anti-ADAMTS-13 antibody comprising, as an activeingredient, a polypeptide or a peptide fragment derived from thepolypeptide composed of the above-described polypeptide or peptidefragment derived from the polypeptide, which lacks reactivity with ananti-ADAMTS-13 antibody by modification such as molecular substitution,deletion, or insertion. The modification such as molecular substitution,deletion, or insertion used herein refers to the deletion, substitution,or addition of one or several amino acids, for example, in an amino acidsequence of the above-described polypeptide or peptide fragment derivedfrom the polypeptide. Such modification allows, for example, alterationin the structure of the polypeptide or the peptide fragment andtherefore allows the loss of the epitope. Accordingly, the polypeptideor the peptide fragment loses reactivity with the anti-ADAMTS-13antibody. When the polypeptide of the present invention recognized by anADAMTS-13 antibody is used, for example, as a neutralizing agent for anautoantibody by administering the polypeptide to a patient positive foran anti-ADAMTS-13 antibody, the polypeptide can be diluted with asaline, a buffer solution, or the like, and made into a preparation toobtain a pharmaceutical composition. The pH of the preparation ispreferably in the pH range from weakly-acidic pH to neutral pH, which isclose to body fluid pH; a lower limit thereof is preferably from pH 5.0to 6.4 and an upper limit thereof is preferably from pH 6.4 to 7.4. Thepreparation can also be provided in a form storable for a long periodsuch as a freeze-dried form. In this case, when used, the preparationcan be dissolved in water, a saline, a buffer solution, or the like, andthen used at a desired concentration. The preparation of the presentinvention may contain pharmacologically acceptable additives (e.g.,carriers, excipients, and diluents) and stabilizers that are usuallyused in drugs or other ingredients that are pharmaceutically required.The stabilizers are exemplified by monosaccharides such as glucose,disaccharides such as saccharose and maltose, sugar alcohols such asmannitol and sorbitol, neutral salts such as sodium chloride, aminoacids such as glycine, nonionic surfactants such as polyethylene glycol,a polyoxyethylene-polyoxypropylene copolymer (Pluronic), andpolyoxyethylene sorbitan fatty acid ester (Tween), and human albumin. Itis preferred that any of these stabilizers on the order of 1 to 10 w/v %should be added to the preparation.

The pharmaceutical composition of the present invention can beadministered in an effective amount by, for example, intravenousinjection, intramuscular injection, or subcutaneous injection and isadministered in a single dosage or several dosages. The dosage variesdepending on the symptom, age, body weight, and so on, of a patient andis preferably 0.001 mg to 100 mg per dosage.

The present specification encompasses contents described in thespecification and/or drawings of JP Patent Application No. 2003-071979that serves as a basis for the priority of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a method of producing a C-terminus deletionmutant for determining an epitope for an antibody;

FIG. 2 is a photograph in which the expression and abundance of theprepared C-terminus deletion mutant has been confirmed by Westernblotting under non-reducing conditions using an anti-FLAG antibody;

FIG. 3 is a photograph in which a region recognized by purified IgGderived from an acquired TTP patient 003 has been confirmed by Westernblotting under non-reducing conditions;

FIG. 4 is a photograph in which a region recognized by purified IgGderived from an acquired TTP patient 004 has been confirmed by Westernblotting under non-reducing conditions;

FIG. 5 is a photograph in which a region recognized by purified IgGderived from an acquired TTP patient 009 has been confirmed by Westernblotting under non-reducing conditions; and

FIG. 6 is a photograph in which a more precise region recognized bypurified IgG derived from the acquired TTP patients has been confirmed.

BEST MODE FOR CARRYING OUT THE INVENTION

Although the present invention will be described hereinafter in detailwith reference to Examples, the present invention is not intended to belimited to these Examples by any means.

EXAMPLES Preparation Example 1 Preparation of C-terminus Deletion Mutantof ADAMTS-13

Expression vectors for full-length and mutant (sequentially lackingdomains from the C terminus) (Fu1427, and T1135X, W1016X, W897X, W808X,W746X, W688X, T581X, Q449X, W387X, and P285X; each numerical valuerepresents the number of amino acid residues from Met encoded by aninitiation codon ATG to a stop codon, and X represents a stop codon)genes described in the previous patent application (JP PatentApplication No. 2002-279924) were utilized to perform transfection usingHela cells in the procedures below. Each mutant location in afull-length sequence is shown in FIG. 1.

At first, 24 hours before transfection, 1-3×10⁵ cells per 35-mm dishwere seeded. On the following day, 10 μl of the Polyamine TransfectionReagent TransIT (manufactured by TAKARA) per 2 μg of each of theexpression vectors was added to 200 μl of a serum-free medium such asOpti-MEM to prepare a complex with DNA according to the instructionsattached to the reagent. The complex was then added dropwise to theabove-described various cells prepared in advance and was incubated for6 hours. Seventy-two hours after the incubation, the medium wascollected. The detection of each appropriately concentrated mutant wasconducted Western blotting using an anti-FLAG-M2 antibody (manufacturedby Kodak) and staining with an anti-mouse IgG-alkaline phosphataseenzyme-labeled antibody system (the result of confirming the expressionof the mutants is shown in FIG. 2).

Example 1 Analysis of Epitope for Antibody in Acquired TTP Patient usingWestern Blotting

IgG fractions (antibody concentration: 2 to 5 mg/ml) were prepared fromplasma samples from acquired TTP patients using a protein A columnaccording to a standard method, and diluted 200-fold to perform Westernblotting. An IgG-alkaline phosphatase-labeled antibody was used as asecondary antibody in a filter, and the filter was stained with aBCIP/NBT substrate to visualize bands (FIGS. 3 to 5). The region therebydetermined which is recognized by the antibody was confirmed to belocated on the C-terminus side from the Q449X, because a region up tothe W688X shows reactivity and a region at the Q449X shows no reactivitywith all of the antibody fractions from the three patients,.

Example 2 Analysis of Precise Epitope for Antibody in Acquired TTPPatient using Western Blotting Based on Principle of CompetitiveInhibition

For narrowing down the more precise location of the epitope recognizedby the neutralizing antibody of the present invention, supernatants fromthe W688X mutant and the full-length wild-type ADAMTS-13 were subjectedto electrophoresis and transferred to a PVDF membrane. The PVDF membranewas utilized in a competitive inhibition system using primary antibodyreaction solutions obtained by previously preincubating theabove-described antibodies from the patients with a concentratedsupernatant having the considerable overexpression of the Q449X mutant,the W688X mutant, or the full-length wild-type ADAMTS-13. As a result,bands positive for the full-length wild type ADAMTS-13 for all of thesamples were confirmed to disappear by the W688X mutant (FIG. 6).

This has suggested that all of the antibodies from the three patientsrecognize a region located on the N-terminus side from the W688X.

From the results shown in Examples 1 and 2, a major neutralizing regionfor the autoantibodies used from the three patients was confirmed to bea region located on the terminus side of the Q449X and on the N-terminusside from the W688X, that is, a region from a Cys-rich region to aspacer region which is between the Q449X and the X688X.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The findings brought about by the present invention demonstrate that apolypeptide of this invention shows specific immunoreactivity with ananti-ADAMTS-13 antibody. Therefore, the use of the polypeptide allowsthe rapid detection of the amount of the anti-ADAMTS-13 antibody,diagnosis for diseases associated with variations in the amount of aprotease of the present invention, or the neutralization of the bindingor inhibitory activity of the anti-ADAMTS-13 antibody. Thus, thepolypeptide provided by the present invention provides a wide variety ofapplications including the detection of the anti-ADAMTS-13 antibody andother applications.

The present invention exhibits such remarkable action or effect and canbe said to be an invention of great significance which contributes toevery field.

1. A polypeptide comprising a neutralizing epitope region in vonWillebrand factor-specific cleaving protease (hereinafter, also referredto as vWFCP or ADAMTS-13), which is recognized by an antibody againstthe protease, or a peptide fragment derived from the polypeptide.
 2. Thepolypeptide or the peptide fragment derived from the polypeptideaccording to claim 1, wherein the neutralizing epitope region is locatedin a region from position 449 to position 687 in an amino acid sequenceshown in SEQ ID No.
 1. 3. A polypeptide comprising an amino acidsequence from position 449 to position 687 in an amino acid sequenceshown in SEQ ID No. 1, or a peptide fragment derived from thepolypeptide.
 4. A polypeptide comprising an amino acid sequenceconsisting of an amino acid sequence from position 449 to position 687in an amino acid sequence shown in SEQ ID No. 1, where one or severalamino acids are deleted, substituted, or added, the polypeptide beingrecognized by an antibody against von Willebrand factor-specificcleaving protease, or a peptide fragment derived from the polypeptide.5. An antibody capable of binding to a polypeptide or a peptide fragmentderived from the polypeptide according to any one of clams 1 to
 4. 6.The antibody according to claim 5, which is present in blood of apatient positive for an anti-ADAMTS-13 antibody.
 7. The antibodyaccording to claim 5 or 6, which is present in blood of a patient withnon-familial thrombocytopenic purpura (hereinafter, also referred to asTTP).
 8. A reagent for antibody measurement comprising a polypeptide,having a complete sequence composing ADAMTS-13, or a polypeptide or apeptide fragment derived from the polypeptide according to any one ofclaims 1 to
 4. 9. The reagent for antibody measurement according toclaim 8, wherein an autoantibody in a TTP patient is an object to bedetected.
 10. A pharmaceutical composition for treating a patientpositive for an anti-ADAMTS-13 antibody comprising, as an activeingredient, a polypeptide or a peptide fragment derived from thepolypeptide according to any one of claims 1 to
 4. 11. Thepharmaceutical composition for treating a patient positive for ananti-ADAMTS-13 antibody according to claim 10, wherein thepharmaceutical composition comprises, as an active ingredient, apolypeptide or a peptide fragment derived from the polypeptide composedof a polypeptide or a peptide fragment derived from the polypeptideaccording to any one of claims 1 to 4, which lacks reactivity with ananti-ADAMTS-13 antibody by modification such as molecular substitution,deletion, or insertion.
 12. The pharmaceutical composition for treatinga patient positive for an anti-ADAMTS-13 antibody according to claim 10or 11, wherein the pharmaceutical composition is administered to thepatient to thereby neutralize the antibody.
 13. A composition comprisinga ligand specific to an anti-ADAMTS-13 antibody for treating a patientpositive for an anti-ADAMTS-13 antibody, comprising, as an activeingredient, a polypeptide or a peptide fragment derived from thepolypeptide according to any one of claims 1 to 4, which is bound with acarrier and brought into contact with plasma from the patient to be usedfor removing the anti-ADAMTS-13 antibody from the plasma from thepatient.
 14. A method of producing blood or plasma free from ananti-ADAMTS 13 antibody by bringing a carrier bound with a polypeptideor a peptide fragment derived from the polypeptide according to any oneof claims 1 to 4 into contact with blood or plasma from a patientpositive for an anti-ADAMTS-13 antibody and binding an anti-ADAMTS-13antibody in the blood or the plasma to the ligand to remove theanti-ADAMTS-13 antibody from the blood or the plasma.